Malate thiokinase catalyzes ATP dependent thiol ester formation between coenzyme A and a number of dicarboxyllic acids. We have found that the acyl CoA product of the reaction remains tightly bound to the enzyme during catalysis. Initially each of the four active sites contains bound acyl-CoA, with half of these displaced by ATP. These results support the hypothesis that malate thiokinase reacts by a mechanism involving half-of-the-site reactivity. An active-site directed sulfhydryl reagent, methoxy-carbonyl CoA disulfide, has been used to probe the acyl-CoA site of malate thiokinase. Inactivation of the enzyme by this reagent appears to involve a specific reaction at this acylCoA site. Enzyme inactive towards turnover, is still active in catalyzing and ATP-ADP exchange reaction. Further studies will be pursued using this reagent to probe the effect of bound acyl-CoA on enzyme turnover.